《出生前基因型》PPT课件.ppt

PrenatalgenotypingusingRFLPThedifferencesinRFLPpatternsareusedtodiagnosegeneticdisordersprenatallyandcanalsodeterminethegenotypeofparentsandotherfamilymemberswhomaybeheterozygouscarriers ForprenataldiagnosisfetalcellsareobtainedbyamniocentesisThetoolsneededtoperformtheseDNAdiagnosesarefirstweneedarestrictionenzymethatwillcutDNAinamannerthatisinformativeforthediseaseinquestion andsecondweneedanucleicacidprobethatwilldetectthatregion1kb4kbsampleA probexxxxxxxxxxSampleB xxxxxxxxxxx ThetwoDNAsamplesarecutwiththeappropriateenzymeandarehybridizedwiththecorrectprobe Theprobehybridizedtothe1and4kbfragmentsinsampleAandthe5kbfragmentinsampleB ThisisanexamplewherearestrictionsitehasbeengainedinsampleA orlostinsampleB bymutation SampleASampleB 5 0kb 4 0kb 1 0kb GenotypeDeterminationusingRFLP sandaGeneProbeADNAprobethathybridizestothe5 endofthehumanbetaglobingene showninblueonthediagrambelow wasusedtoidentifyRFLPpiecesfrommembersofafamilyinwhichsickle cellhemoglobin HbS wassegregating ThenormalHBallele HbA iscutatthreeplacesbyaparticularrestrictionenzyme positionsshownwithredarrows TheHbSmutationdestroystheinternalrestrictionsitesotheHbSgeneiscutinonlytwoplaces Thus theprobehybridizestoa1 15kbDNAfragmentfromHbADNAandhybridizestoa1 35kbfragmentfromHbSDNA LinkedDNAmarkersAlloftheDNA RFLPorPCR markersthatwehavediscussedsofarhavebeentargetedtoaspecificgene Formanyimportanttraits theactualgeneofinterestisnotknown Therefore probingforthepresenceofthenormalormutatedalleleisnotpossible Insteadtheproberecognizesasequencethatisclosetotheactualgeneofinterest BoththephenotypeandtheRFLPmarkerisfollowedinthegenerations RecombinationfrequenciescanbeestimatedandthedistancebetweenthemarkerandthegeneofinterestcanbedeterminedMarker1Marker21cM5cMGeneMarker1GeneMarker2 There sonly1 chancethatthelinkageisbroken withmarker1 DNAsequencing IsthedeterminationoftheprecisesequenceofnucleotidesinasampleofDNA Themostpopularmethodfordoingthisiscalledthedideoxymethod chainterminationmethod DNAissynthesizedfromfourdeoxynucleotidetriphosphates dNTP Eachnewnucleotideisaddedtothe3 OHgroupofthelastnucleotideaddedinthepresenceofDNApolymerase TheDNAtobesequencedispreparedasasinglestrand ThistemplateDNAissuppliedwithaprimerhavingcomplementarysequence DNAsampleandtheprimerisseparatedinto4tubesandamixtureofall4 deoxy nucleotideswhichareradiolabelledareaddedinamplequantitiestoall4tubesplusDNApolymeraseI Onedideoxynucleotideisthenaddedinaverysmallquantitytoeachtube Becauseallfournormalnucleotidesarepresent chainelongationproceedsnormallyuntil bychance DNApolymeraseinsertsadideoxynucleotide Attheendoftheincubationperiod thefragmentsareseparatedbylengthfromlongesttoshortest Theresolutionissogoodthatadifferenceofonenucleotideisenoughtoseparatethatstrandfromthenextshorterandnextlongerstrand Thefragmentsfromeachreactiontubeareseparatedinfouradjacentlanesbygelelectrophoresis Fig16 13 1 YourecoveraclonedDNAsegmentofinterest anddeterminethattheinsertis1300bpinlength Tocharacterizethisclonedsegmentyouisolatetheinsertanddecidetoconstructarestrictionmap DigestingwithenzymesEcoRIandBamHIandfollowingelectrophoresis youdeterminethenumberandsizeofthefragmentsproducedbyEcoRIandBamHIaloneandincombinationasfollows EnzymeRes FragmentsizesEcoRI350bp 950BamHI200bp 1100bpEcoRI BamHI150bp 200bp950bp 350EcoRI950200150BamHI 2 Aninbredstrainofplantshasameanheightof24cm Asecondstrainofthesamespeciesfromadifferentcountryalsohasameanheightof24cm TheF1plantsfromacrossbetweenthesetwostrainsarealso24cmhigh However theF2generationshowsawiderangeofheights themajorityareliketheP1andF1plants butapproximately4of1000areonly12cmhigh and4of1000are36cmhigh i Whatmodeofinheritanceisoccurringhere ii Howmanygenepairsareinvolved iii Howmuchdoeseachgenecontributetotheplantheight iv IndicateonepossiblesetofgenotypesoftheP1andF1plantsthatcouldexplaintheirheights v IndicateonepossiblesetofgenotypestoaccountforF2plantsthatare18cmor33cmhigh i Whatmodeofinheritanceisoccurringhere Polygenicinheritancewhereacontinuoustraitisinvolvedandallelescontributeadditivelytothephenotypeisdemonstrated ii Howmanygenepairsareinvolved 4 1000 1 250 1 4n n 4 iii Howmuchdoeseachgenecontributetotheplantheight 36 12 24 8 3 iv IndicateonepossiblesetofgenotypesoftheP1andF1plantsthatcouldexplaintheirheights Iftheshortestisaabbccdd isthebaseheightthen24 12 12 4additiveallelesshouldbeinbothP1andF1 AABBccddxaabbCCDDAaBbCcDd v IndicateonepossiblesetofgenotypestoaccountforF2plantsthatare18cmor33cmhigh Forplantwith18 18 12 6onlytwoadditiveallelesAaBbccdd etcForplantwith33 33 12 21only7additiveallelesAABBCCDdetc 3 Trihybrideggplantswithhairyleaves purpleflowers andthornystalksaretestcrossedwithplantshavinghairlessleaves whiteflowers andsmoothstalks Theprogenyare PhenotypeNumberhairy purple thorny10hairy purple smooth132hairy white thorny81hairy white smooth998hairless purple thorny1020hairless purple smooth101hairless white thorny150hairless white smooth8Deriveageneticmapofthethreeloci Whatisthecoefficientofcoincidence hairy purple thorny10hairy purple smooth132hairy white thorny81hairy white smooth998 wsw sws hairless purple thorny1020h h hhairless purple smooth101incorcorrincorhairless white thorny150hairless white smooth8total2500Bet Handw 150 132 10 8 2500 0 12 12muBetHands 81 101 10 8 2500 0 08 8muObserveddco 18 2500 0 007Expdco 0 12x0 08 0 009c 0 007 0 009 0 77 Squiggly eyedmaleflieswerecrossedwithnormalfemales andtheresultingF1progenywere 122normalmales131squiggly eyedfemalesWhatisthemodeofinheritanceforsquigglyeyes Whatphenotyp